Normal human bronchial epithelial cells are cultured in a defined, serum-free medium. We investigated the mitogenic effects of compounds using clonal growth assays and colony-forming efficiency. We also measured ornithine decarboxylase activity and cAMP levels, since polyamine and cAMP metabolism are involved in cell proliferation in most systems. In the defined medium LHC-O, which already contains insulin, hydrocortisone, transferrin, phosphoethanolamine, and ethanolamine, the cells grow at a rate of 0.42 population doublings per day (PD/D). Many compounds were investigated, and most had no mitogenic effect, including human pituitary growth hormone, testosterone, estridiol, estriol, calcitonin, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and endothelial cell growth supplement (ECGS). EGF, bombesin, gastrin-releasing peptide amino acids 14-27 (GRP14-27), and human chorionic gonadotropin (HCG) each independently increase the clonal growth rate of 0.68, 0.65, 0.67, and 0.66 respectively. An aqueous extract of bovine pituitaries (PEX) has no effect by itself but does increase the clonal growth rate when EGF is also present. cAMP-enhancing agents are mitogenic only when both EGF and PEX are present. When bombesin, 0.1 micromolar is present, the concentration of PEX required to provide a half maximal growth respose is decreased from 2.27 micrograms/ml to 0.4 micrograms/ml.